Method for producing human monoclonal antibodies that binds to at least one part of HMGB1

ABSTRACT

The present invention relates to methods of making human anti-HMGB1 antibodies, compositions comprising these antibodies and methods of using the antibodies and compositions in HMGB1 associated-neuropathy.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of filing date of U.S. ProvisionalApplication Ser. No. 61/899,473, entitled “Complete human neutralizingantibody for high mobility group box 1 (HMGB1)-associated neuromyelitis”filed Nov. 4, 2013, the entire contents of which are specificallyincorporated herein by reference.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates to neutralizing antibodies targeting highmobility group box 1 (HMGB1), and compositions and methods comprisingsuch antibodies useful in treating, inhibiting or preventingHMGB1-associated neuropathy. The invention also relates to thecombination of the variable regions of heavy and light chainimmunoglobulins derived from a single human antibody-producing cell andamino acid molecules encoding such antibodies and antigen-bindingportions, methods of making human anti-HMGB1 antibodies andantigen-binding portions, compositions comprising these antibodies andantigen-binding portions and methods of using the antibodies,antigen-binding portions, and compositions.

2. Description of Related Art

In normal physiological conditions, HMGB1 is confined to the nucleus asa non-histone DNA binding protein that is involved in regulation ofchromatin dynamics (Hock et al., 2007), such as stabilizing nucleosomeformation (Goodwin et al., 1973), facilitating gene transcription(Kleinschmidt et al., 1983) and modulating steroid hormone receptors(Boonyaratanakornkit et al., 1998). In contrast, in the case of tissueinjury, HMGB1 migrates toward the cytoplasm and then is releasedextracellularly, acting as a danger signal (Bianchi and Manfredi, 2007).In addition to being passively released by necrotic cells, HMGB1 canalso be actively secreted by specific cells, e.g., macrophages andmyeloid cells, of the innate immune system activated by pro-inflammatorysignals such as lipopolysaccharide, tumor necrosis factor-α (TNF-α) andinterleukin-1 β (IL1-β) (Bonaldi et al., 2003). It has been alsodemonstrated as a cytokine that mediates endotoxin lethality (Wang etal., 1999), propagating inflammation (Wang et al., 2001), prolongingmacrophage activation (Andersson et al., 2000) and chemoattractingmesoangioblast (Palumbo et al., 2004). It is becoming increasingly clearthat both passively released and actively secreted extracellular HMGB1convey the same danger signal to attract and activate inflammatory cells(Abraham et al., 2000; Andersson et al., 2000), to enhance theexpression of cell adhesion molecules in endothelial cells (Fiuza etal., 2003) and to impair the barrier function of endothelium (Yang etal., 2006). Specifically, serum levels of HMGB1 have been directlyassociated with mortality in patients with lethal sepsis and inexperimental models of endotoxaemia (Wang et al., 1999; Fink, 2007),suggesting that HMGB1 is a crucial late mediator of the sustainedactivation of pro-inflammatory cascades associated with fatal outcomes.Specific receptors interacting with extracellular HMGB1 include receptorfor advanced glycation end products (RAGE), toll-like receptor (TLR)-2and TLR-4 (Hori et al., 1995; Park et al., 2004). The binding of HMGB1to its receptors results in the activation of several kinases such asextracellular signal-regulated kinases (ERK)-1/2, p38 mitogen-activatedprotein (MAP) kinases, and c-Jun NH2-terminal kinase, which ultimatelyleads to the activation of nuclear factor-κB (NF-κB)-dependent genes(Huttunen et al., 1999; Fiuza et al., 2003).

As human HMGB1 has cytokine activities and mediates prolonged systematicinflammation as well as immune responses, we have thus investigated andsuccessfully established the link between an aberrant production ofHMGB1 in plasma of patients with neuromyelitis, i.e., neuromyelitisoptica (NMO) and conventional multiple sclerosis (MS) (Wang et al.,2012). NMO and MS are a chronic, inflammatory, demyelinating disease ofthe central nervous system (CNS), most frequently starting with a seriesof bouts, each followed by complete remission and then a secondary,progressive phase during which the neurological deficit increasessteadily. However, the clinical course of NMO is usually more severethan classical or conventional MS (Cornelio et al., 2009). Within fiveyears of onset, fifty percent of NMO patients either loss of functionalvision in at least one eye or becoming unable to walk unassisted, or theannual costs for hospitalization exceed several hundred million. Thereremains an urgent need for prophylactic, preemptive and treatmentstrategies for management of HMGB1-associated neuromyelitis.

Anti-huHMGB1 therapies by using of monoclonal antibodies (mAbs) tospecifically bind to and neutralize this particular target protein areexpected to be effective in nature. Accordingly, targets with highlyselective late mediator are preferred to broaden the therapeutic window(Wang et al., 1999). Furthermore, given the remarkable divergentemergence of the physiological from pathological conditions of huHMGB1,mAbs raised against it are expected to selectively target extracellularHMGB1. Based on these considerations, a monoclonal antibody againsthuHMGB1 that may inhibit receptor interaction and therefore block kinaseactivation and the downstream pro-inflammatory response, e.g., TNF-αsecretion is highly desirable.

Although mAbs are long-established as essential research tools, theirtherapeutic promise has taken considerably longer to realize, requiringfurther advances, such as the humanization of mouse antibodies and/ortransgenic protocols, to reduce their immunogenicity inherent in murineproteins. The biopharmaceutical industry has thus seen a full shift ofnew antibody therapeutics from chimeric to humanized to fully-humansequences. The ultimate objective in the industry is to manufacture amAb drug that is identical to that which is produced in the human body.

In theory, heavy and light chain components derived entirely from thehuman origin could be used to assemble mAbs. Indeed, the use of phageantibody technology to construct human heavy and light chain librariesas described in e.g., WO92/01047, U.S. Pat. No. 5,652,138 and U.S. Pat.No. 5,885,793A, offers the ability to isolate such human antibodiesdirectly. Moreover, U.S. Pat. No. 5,652,138 and U.S. Pat. No. 6,075,181demonstrate the feasibility to construct in vivo transgenes to introduceinto a non-human animal substantially the entire human immunoglobulinloci and subsequently perform animal immunization to obtainheavy-and-light-chain pairs comprised human sequences. However, theresultant pairings may not be naturally-occurring and still remain to beimmunogenic as indicated in the official FDA labeling information(www.accessdata.fda.gov)—the frequencies of patient to generateanti-drug antibodies can be as high as 26% and 3.8% for Humira(adalimumab) and Vectibix (panitumumab), respectively. Therefore, thismay not be the best available strategy. In fact, while there are generalrules (De Groot and Scott, 2007), predicting the precise immunogenicityof a specific protein in the genetically heterogeneous human populationis a difficult if not a fundamentally impossible task. This ishighlighted again by the case of Enbrel (etanercept), which is abiopharmaceutical produced by recombinant fusion between the TNFreceptor and the constant region of the IgG1 antibody to act as a TNFinhibitor. Since both fusion partners are bona fide naturally-occurringubiquitous proteins in humans and, in principle, should not beimmunogenic. Yet clinical data specify that 5.6% treated patients testedpositive for anti-etanercept antibodies (Dore et al., 2007).

Our understanding in all of the area of immunogenicity is increasing,but currently it seems impossible to close the circle. Alternatively, asharp clinical contrast provided by the therapeutic immunoglobulinpreparations, and especially intravenous immunoglobulin (IVIg), may wellturn out to be optimistic in resolving the problem of immunogenicity.The IVIg refers to a therapeutic biological product containing human IgGthat is prepared by large-scale industrial fractionation of human plasmaderive from samples collected from thousands of blood donors(Kazatchkine and Kaveri, 2001; Seite et al., 2008), and thus representsin essence a preparation of human polyclonal antibodies. It usuallyadministrated 400 milligrams to 1 gram per kilogram body weight weekly,by which it generally acts as a replacement therapy for many years(Farrugia and Poulis, 2001; Wittstock et al., 2003). Despite theworldwide consumption of IVIg may well above 100 tonnes per year,adverse reactions to IVIg occur in less than five percent of patients(consensus.nih.gov/1990/1990IntravenousImmunoglobulin080html.htm), alsopatients seem to tolerate to IVIg and very few if there is any anti-IVIgresponses reported (Lemieux et al., 2005; Imbach, 2012b; Imbach, 2012a).Lack of immunity to IVIg suggests that ingenious designs to harnessnaturally-occurring configurations inherent in a singleantibody-producing B cell could provide a substitute for such approach.

Nevertheless, while foreign antigens, which are molecules derived from apotentially harmful invader, trigger the production of antibodies by theimmune system; self antigens such as HMGB1 are usually tolerated by theimmune system. In reality, although self antigen-reactive B cells may bepresent in the body, self antigens are not likely to initiate an immuneresponse and thus without leading to the production of specificantibodies. To effectively transform such challenging circumstances,“site-directed in vitro immunization” (Chin et al., 2007) (U.S. Pat.Nos. 7,494,779, 8,021,860 and 8,158,386) was developed by the inventorsto initiate in vitro self antigen-specific immune responses from primaryhuman lymphoid cells. Self antigens can then be neutralized by theresultant mAbs similar to foreign ones.

One of the major problems in obtaining highly potent human therapeuticmAbs is how to isolate antigen-specific clones derived from thesite-directed in vitro immunization scheme or even the circulatingmemory B cell compartment of a human donor. This so-called rarepopulation are clones that under-representative and, accordingly, verylittle in number. Some of these rare specific cells are expected to bemost interesting in modern medicine and thus much research has attemptedto address this question, including the use of the antigen-conjugatedchitosan as taught by WO 2010104828. However, in light of thelimitations of pH-dependent solubility and the reactivity of its aminegroups in enzymatic conjugation inherited in biopolymer chitosan toaccount for many aspects of protein conjugation, it will clearly beimportant to develop a method for the separation of immune cells byinteractions of proteins of said B cells with antigens.

Rabbit antisera have been generated against a synthetic peptidecorresponding to the amino terminal part of HMGB1 (amino acid residues2-15), coupled to radially branching lysine dendrites and against theentire HMGB1 (U.S. Pat. No. 6,468,533). Murine mAbs produced from ahybridoma resulting from the fusion of mouse myeloma with B cellsobtained from a mouse immunized with a part of huHMGB1 (amino acidresidues 89-1162) or purified recombinant rat HMGB1 (U.S. Pat. No.7,288,250). Rat mAbs specific to the carboxyl terminal (amino acidresidues 200-211) were reported using HMGB1/HMGB2 as an immunogen (Liuet al., 2007). R&D Systems, Inc. makes and sells a polyclonal anti-humanHMGB1 antibody (Cat. # AF 1690) produced in chicken immunized withpurified, NS0-derived recombinant human HMGB1.

To date, no complete human mAbs with naturally-occurring configurationsinherent in a single antibody-producing B cell to HMGB1 neither have theuse of such kind of mAbs for inhibiting or treating HMGB1-associatedneuromyelitis been reported.

SUMMARY OF THE INVENTION

The object of the present invention is to provide neutralizingantibodies targeting high mobility group box 1 (HMGB1), specifically,the neutralizing antibodies not only containing exclusively humansequences but also comprising naturally-occurring configuration ofheavy-and-light-chain combination.

Another object of the present invention is to provide an isolatedcomplete human neutralizing anti-HMGB1 mAbs or antigen-binding portionsthereof that bind to HMGB1, preferably the neutralizing epitopes ofHMGB1, more preferably the HMG A box and RAGE binding segment of HMGB1.In a preferred embodiment, the neutralizing antibodies are completehuman monoclonal antibodies or antigen-binding portions thereof.

Another object of the present invention is to provide a method forproducing complete human neutralizing anti-HMGB1 mAbs or antigen-bindingportions thereof that binds to at least one part of HMGB1, and theresultant mAbs produced by the method target specific domain of HMGB1protein with less allergic potential.

Another object of the present invention is to provide a method forinhibiting or treating HMGB1-associated neuromyelitis, comprisingadministering an effective amount of complete human neutralizinganti-HMGB1 mAbs to a subject in need thereof, the complete humanneutralizing anti-HMGB1 mAbs effectively inhibit and/or treatHMGB1-associated neuromyelitis with less allergic responses.

To achieve the objects, the present invention provides an artificialneutralizing antibodies targeting high mobility group box 1 (HMGB1). Theartificial neutralizing antibodies is preferably an complete humanneutralizing anti-HMGB1 mAbs and produced by the following steps of: (a)providing a synthetic antigen comprising at least one peptide havingamino acid sequence represented by SEQ ID NO:3 and SEQ ID NO:4 toimmunize a group of human CD56⁻ lymphocytes; (b) the lymphocytesimmunized by the synthetic antigen are enriched by antigen-coupledmagnetic beads; (c) adding Epstein-Barr virus (EBV) to the human CD56⁻lymphocytes immunized by the synthetic antigen; and (d) selecting theEBV-infected cells that produce the antibodies that recognize HMGB1.

The present invention provides a method for producing an complete humanneutralizing anti-HMGB1 mAbs or antigen-binding portions thereof thatbinds to at least one part of HMGB1, comprising the following steps of:(a) providing a synthetic antigen comprising at least one peptide havingamino acid sequence represented by SEQ ID NO:3 and SEQ ID NO:4 toimmunize a group of human CD56⁻ lymphocytes; (b) the lymphocytesimmunized by the synthetic antigen are enriched by antigen-coupledmagnetic beads; (c) adding Epstein-Barr virus (EBV) to the human CD56⁻lymphocytes immunized by the synthetic antigen; and (d) selecting theEBV-infected cells that produce the antibodies that recognize HMGB1.

In a preferred embodiment of the present invention, in step (b), thelymphocytes immunized by the synthetic antigen are enriched byantigen-coupled magnetic beads by the following steps: (a) His-taggedHMGB1 proteins are immobilized on magnetic beads to form HMGB1-beadscomplex; (b) mixing the HMGB1-beads complex and the lymphocytes; and (c)releasing the lymphocytes that trapped by the HMGB1-beads complex.Specifically, the “HMGB1 proteins” that immobilized on magnetic beadsmay be a full-length HMGB1 protein or a specific domain of a HMGB1protein, such as HMG A box or RAGE binding segment of HMGB1.

In addition, the present invention also provides a method for inhibitingor treating HMGB1-associated neuromyelitis, comprising administering aneffective amount of a human monoclonal antibodies to a subject in needthereof, wherein the human monoclonal antibodies are produced by thesteps of: (a) providing a synthetic antigen comprising at least onepeptide having amino acid sequence represented by SEQ ID NO:3 and SEQ IDNO:4 to immunize a group of human CD56⁻ lymphocytes; (b) the lymphocytesimmunized by the synthetic antigen are enriched by antigen-coupledmagnetic beads; (c) adding Epstein-Barr virus (EBV) to the human CD56⁻lymphocytes immunized by the synthetic antigen; and (d) selecting theEBV-infected cells that produce the antibodies that recognize HMGB1.

Other objects, advantages, and novel features of the invention willbecome more apparent from the following detailed description when takenin conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a representative ELISA reactivity profile of culturesupernatant. Diluted supernatants were tested in duplicate with 100 μLadded to each well. Deviation between duplicate was less than 10% forany reported value;

FIG. 2 is a histogram depicting the representing results of HMGB1neutralizing experiments of two human mAbs. Human TNF was induced bystimulating monocytes with 1 μg/ml of bovine HMGB1. Purified HMGB1monoclonal antibodies were added to give a final concentration of 10%;

FIG. 3 shows specific antigen-antibody reaction was detected on thesurface of a QCM chip. Real-time monitoring of protease reaction denotedthat competition was observed when the peptide used containing theimmunogen sequence but not amino acid composition-compatible peptide,indicating the specificity of mAb towards the corresponding immunogen;and

FIG. 4 denotes anti-HMGB1 contributes to EAE alleviation in experimentalanimals. Data are representative of two experiments of ≥5 mice pergroup.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Prior to describing the invention in further detail, the terms used inthis application are defined as follow unless otherwise indicated.

The term “naturally-occurring”, as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, acombination of heavy and light chain germline immunoglobulins derivedfrom a single human antibody-producing (B) cell that is present in ahuman that can be isolated from a source in nature, e.g., peripheralblood, and which has not been intentionally modified by man in thelaboratory is naturally-occurring.

The term “monoclonal antibody (mAb)” as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that maybe present in minor amounts. Monoclonal antibodies are highly specific,being directed against a single antigenic site (epitope). The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod.

The term “complete human antibody”, as used herein with regard to anantibody, means that is an antibody not only containing exclusivelyhuman sequences but also comprising naturally-occurring configuration ofheavy-and-light-chain combination. The antibody is preferably amonoclonal antibody.

“Non-immunogenic in a human” means that upon contacting the polypeptideof interest in a physiologically acceptable carrier and in atherapeutically effective amount with the appropriate tissue of a human,no state of sensitivity or resistance to the polypeptide of interest isdemonstrable upon the second administration of the polypeptide ofinterest after an appropriate latent period e.g., 8 to 14 days. It willbe understood by one of skill in the art that a polypeptide of completehuman origin or a polypeptide combination with a naturally-occurringgermline configuration typically represents “non-immunogenic in ahuman”.

As used herein, the term “site-directed in vitro immunization” refers toin vitro antigen-specific immune responses from primary human lymphoidcells (Chin et al., 2007) (U.S. Pat. Nos. 7,494,779, 8,021,860 and8,158,386). This technology relies on presence of coupled T- and B-cellepitopes as well as depleted starting lymphoid populations containingprimed precursors recognizing T-cell epitope.

A “heteromyeloma cell line” is a cell line derived from fusion of twodifferent myeloma cells. The two different myeloma cells are preferablya human myeloma cell and a murine myeloma cell. Heteromyeloma cell linesare known in the art. For example, U.S. Pat. No. 6,228,361 and Chin etal., 2001 describe the preparation, characterization and use of variousheteromyeloma cell lines.

A “fusion partner” is a cell that can be used to fuse with anantibody-producing cell for a beneficial purpose. Typically, the fusionleads to prolonged antibody production. Thus, without fusion to thefusion partner, the antibody-producing cell ceases to produce antibodiesin culture. Upon fusion to the fusion partner, however, fused cells canbe selected that produce antibodies in culture for at least about 3months, preferably at least about 6, 9, 12, 18, 24 months or more.Fusion partners include, but are not limited to, myeloma cells andheteromyeloma cells.

As used herein, the term “antigen-binding region” refers to that portionof an antibody molecule which contains the amino acid residues thatinteract with an antigen and confer on the antibody its specificity andaffinity for the antigen. The antigen-binding region typically comprisesthe amino acid residues contributed from the paring of the correspondingheavy and light chains necessary to maintain the proper conformation ofthe antigen-binding residues. The peculiar configuration of specificparing is inherent in a particular antibody-producing B cell.

The term “original (VH/VL) pairing” has been used in the art and hereinto illustrate that although a significant number of the available heavyand light chain germline gene segments are used in VH/VL pairing and agiven VH sequence can pair with many light chain sequences of bothlambda and kappa light chain classes in human Ab generation, the VH/VLpairing is unique in a given B cell survived from peripheral selection.The VH/VL interface between the light chain and heavy chain has beenshown to affect the binding kinetics of a peptide (Chatellier et al.,1996), suggesting preference for particular pairings. The interactionsbetween VH/VL contribute significantly to the stability of theantigen-binding region as limited structural changes have significantfunctional effects on binding affinity. The translation of the humanimmunoglobulin germline sequences including all known alleles has beenprovided online such as www.imgt.org/.

The present invention is based on the ingenious designs the coupled T-and B-cell epitopes to harness naturally-occurring configurationsinherent in a single antibody-producing B cell and simultaneously toisolate antibody-producing B cells with specificity against a selfantigen, i.e., HMGB1 (SEQ ID NO:1). It is a very demanding task becauseof a phenomenon called tolerance; healthy human peripheral lymphoidcells do not normally produce antibodies to self antigens. However,because the site-directed in vitro immunization system utilizes apre-existing helper T cells derived from tetanus immunization to driveB-cell maturation, several advantages are realized over conventionalhuman mAb strategies. First, the protocol yields antigen-specific Bcells rather than antigen-reactive Ig genes, thus eliminating the needof random, non-naturally-occurring recombination required to achieve adetectible Ig binder like in phage display and Ig transgeneintroduction. Secondly, the rate at which newly formed B cells enter themature peripheral pool accounts for less than 5% of the immature cellsgenerated in the bone marrow, suggesting a significant selectionpressure (Cancro, 2004) and thus a possible mechanism to avoidimmunogenicity. The in vivo selection cycles help to circumvent theproblems associated with immunogenicity, a process also taken by IVIg toestablish a general non-immunogenic status in humans. Third, using an invitro stimulation scheme, greater than normal antibody repertoire can beobtained from a population of lymphoid cells after antigen exposure.This system is particular useful for the isolation of complete humanantibodies against the self antigens.

Culture materials and reagents are known in the art and may be obtainedcommercially. The culture medium used is RPMI-1640 (HyClone, Logan,Utah), supplemented with 1×non-essential amino acids (Life Technologies,Gaithersburg, Md.), 10% fetal bovine serum (FBS; Life Technologies) and50 μg/ml of gentamycin and kanamycin (Sinton Chemical & Pharmaceutical,Hsinchu, Taiwan). Purified bovine HMGB1 (Chondrex Inc, Redmond, Wash.)and recombinant human HMGB1 (Sigma, St. Louis, Mo.) are used inantigen-specific and competing enzyme-linked immunosorbent assay(ELISA), together with peroxidase-labeled goat antibodies against humanIgG and IgM (Zymed Laboratories, South San Francisco, Calif.) or avidinhorseradish peroxidase (eBioscience™, San Diego, Calif.) as thereporting system.

Complete human mAbs are produced from in vitro stimulation and culturetechniques. Generally, plasma and buffy coat samples from healthyroutine blood donors, screened negative for HIV-1/2, HTLV-I/II, HCV,HBsAg and containing normal levels of alanine transferase (ALT), can beobtained from local Blood Centers. Peripheral blood mononuclear cells(PBMC) are isolated by density centrifugation on Ficoll-Paque (GEHealthcare Bio-Sciences™, Uppsala, Sweden) as described elsewhere. Theresulting PBMC are magnetically labeled with CD45RO MACS® microbeads(Miltenyi) then separated by a VarioMACS™ (Miltenyi) instrumentaccording to the manufacturer's instructions. The purified CD45RO⁺Tcells are cultured at a density of 2×10⁶ cells/ml in the culture mediumsupplemented with 50 μM 2-mercaptoethanol and 10 μg/ml pokeweed mitogen(PWM; Sigma™, St. Louis, Mo.). After 24 h, cells are removed by 400×gcentrifugation to collect CD45RO⁺ T cell replacing factor. Removal ofcytotoxic cell populations is similarly performed by using colloidalsuper-paramagnetic microbeads conjugated to monoclonal anti-human CD8and anti-CD56 antibodies (Miltenyi™). Removal of IL-10-producing cellsmay be achieved by using rat anti-human IL-10 (SouthernBiotech™,Birmingham, Ala.) and goat anti-rat IgG microbeads (Miltenyi™).

Site-directed in vitro immunization is performed by using cytotoxiccell-depleted PBMC based on a two-step principle. Primary immunizationis performed by incubating the cells for 6 days in a medium containing10 nM of the heterotopic peptide antigen having amino acid sequencerepresented by SEQ ID NO:5 (QYIKANSKFIGITEL(T-helper epitope of SEQ IDNO:2)-HMG A box (DKARYEREMKTY) (SEQ ID NO:3); Yao-Hong BiotechnologyInc., New Taipei City, Taiwan) and SEQ ID NO:6 (QYIKANSKFIGITEL(T-helperepitope of SEQ ID NO:2)-RAGE binding segment (KDIAAYRAKGKP) (SEQ IDNO:4); Yao-Hong Biotechnology Inc., New Taipei City, Taiwan), 50 μM2-mercaptoethanol, 10% heat-inactivated human serum, 0.05 ng/mlrecombinant human (rh) IL-2 (eBioscience™), and 25% (v/v) CD45RO⁺ T cellreplacing factor. For secondary immunization, 3×10⁷ primary-immunizedcells are mixed with the peptide in a flask that had been immobilizedovernight with 5 mg/ml of CD40L (CD154; eBioscience™) together with1×10⁷ SEQ ID NO:2 (QYIKANSKFIGITEL) (Yao-Hong BiotechnologyInc.™)-stimulated CD4⁺ T cells and 5 ng/ml rh IL-15 (eBioscience™). Thecells are cultured for 3-5 days in a medium supplemented with 5% humanserum, 50 mM 2-mercaptoethanol and 10 nM heterotopic peptide antigens.

The concept of using immobilized-metal affinity chromatography (IMAC) tofractionate or purify proteins has first been formulated and itsfeasibility shown by Porath et al. in 1975. The idea was based on theknown affinity of transition metal ions such as Zn²⁺, Cu²⁺, Ni²⁺, andCo²⁺ to histidine and then strongly fixed metal ions to a support forfractionating protein containing such an amino acid (His-tag). Thechelating reagents used to fix the metal to a solid support arecommercially available and usually involve a tridentate chelatoriminodiacetic acid (IDA), tetradentate chelators nitrilotriacetic acid(NTA) and carboxymethyl aspartate (CM-Asp). Bound proteins can be elutedand thus fractionated or purified in four following ways: (1) lower thepH; (2) add excess imidazole; (3) add EDTA to remove metal ion frompurification resin and/or (4) add enzymes to cleave His-tag(Gaberc-Porekar and Menart, 2005). It is now well established that IMACis a highly efficient procedure to purify His-tagged proteins; in fact,recombinant proteins are artificially terminally labeled with His tagsin a form of oligo-His to serve as an affinity handle for the ease ofsubsequent purification because naturally occurring proteins are lack ofstretches of continuous His residues. Therefore, unlike biopolymerchitosan which depends on pH and amine groups for conjugation, virtuallyany protein can be purified from a cell extract in just one or twosteps. In one embodiment, the present invention provides a novel methodfor the separation of specific B cells by interactions of proteins ofsaid immune cells with antigens. Typically, the His-tagged HMGB1proteins (Sino Biological™, Beijing, China) are immobilized on NTA⁻Ni²⁺magnetic nanobeads (Taiwan Advanced Nanotech, Taoyuan, Taiwan) and theHMGB1-nanobeads complex is immersed to trap the immune B cells to thecomplex. Specific B cells retained on a magnetic field were thenreleased by citric acid —Na₂HPO₄ buffer containing 1 U/ml thrombin, 1 mMEDTA and 500 mM imidazole (Sigma™).

Subsequently, the in vitro immunized cells are infected withEpstein-Barr virus (EBV) by virus-containing supernatant derived fromthe EBV-producing marmoset cell line B95-8 (American Type CultureCollection, ATCC CRL 1612). The infected cells are seeded at 10⁵/well in96-well plates together with mytomycin (Kyowa Hakko Kogyo, Tokyo,Japan)-treated PBMC as feeder cells (10⁴/well) for the establishment oflymphoblastoid cells and screened for Ab production by ELISA.HMGB1-specific ELISA can be performed by coating 0.25 μg/ml purifiedHMGB1, 1 μg/ml bovine serum albumin (BSA; Sigma) or 1 μg/ml tetanustoxoid (TT; ADImmune™, Taichung, Taiwan) onto microtitre platesovernight at 4° C. Culture supernatants are diluted to the desired levelin 10 mM sodium phosphate buffer (pH 8.0), containing 0.5 M sodiumchloride and 0.1% Tween-20. Coated plates are incubated with dilutedculture supernatants, washed, incubated with peroxidase-labeled goatantibodies against human IgG and IgM and developed (15 min) by additionof 100 μl of the chromogenic substrate o-phenylaenediamine (OPD)(Sigma™). The reaction is stopped after 30 min by adding 1 M sulphuricacid, and the absorbances are read at 490 nm.

Somatic cell hybridization can be further applied to optimize humanantibody and can be generated by electrofusion. Briefly, HMGB1-specificEBV-infected lymphoblastoid cells were fused with heteromyeloma cells(Chin et al., 2001) in an isotonic medium (280 mM sorbitol, 0.5 mMmagnesium acetate, 0.1 mM calcium acetate and 1 mg/ml BSA; pH6.9-7.1).Cell fusion can be induced by high-voltage pulses using a BTX ElectroCell Manipulator ECM 2001 (Harvard Apparatus™, Holliston, Mass.).HMGB1-specific hybrids were selected and cloned by limiting dilution.

Methods for determining whether an antibody or antigen-binding fragmentinhibits a HMGB1-induced pro-inflammatory condition are known to oneskilled in the art. For example, inhibition of the release of aninflammatory cytokine from a cell in culture can be measuredaccordingly. For example, as described and exemplified herein, TNFrelease from human monocytic populations can be measured using CD14⁺cells sorted magnetically with MACS® microbeads (Miltenyi™). TNF that isreleased from the monocytes is measured by a commercial kit obtainedfrom R & D Systems (Minneapolis, Minn.). Methods for measuring releaseof other related cytokines from cells are also known in the art.

Assays to determine affinity and specificity of binding are known in theart, including competitive and non-competitive assays. A competitiveassay is preferred in this analysis. The affinity of the mAb can bedetermined against HMGB1 protein with an AffinixQN quartz crystalmicrobalance (QCM) biosensor (Inishiamu Inc.™, Tokyo, Japan) accordingto the manufacturer's instructions. Different amounts of syntheticpeptides can then be incorporated into the reaction for competition toconfirm the specificity of human mAbs.

The exact pathogenic process to neuropathic diseases is currentlyunknown, but experimental autoimmune/allergic encephalomyelitis (EAE) isprimarily used as an animal model of autoimmune inflammatory diseases ofthe CNS and in many aspects resembles human neuropathic diseases, suchas MS and NMO. This demyelinating model can be used to evaluate theeffectiveness of human mAbs in alleviating diseases and is known to oneskilled in the art. Immunization with myelin oligodendrocyteglycoprotein (MOG) induces a chronic form of EAE. Different amounts ofsynthetic peptides can then be incorporated into the reaction forcompetition to confirm the specificity of human mAbs. Human mAbs canthen be incorporated into the system for verifying the tendency toresolve inflammation and demyelination.

The following examples are offered to illustrate this invention and arenot to be construed in any way as limiting the scope of the presentinvention.

EXAMPLES Example 1. Specificity of Hybrids Cell Line

Ag-specific enzyme-linked immunosorbent assay (ELISA) was performed bycoating 1 μg/ml purified bovine HMGB1, 1 μg/ml recombinant human HMGB1,1 μg/ml bovine serum albumin (BSA; Sigma) or 1 μg/ml tetanus toxoid (TT;ADImmune™, Taichung, Taiwan) onto microtitre plates overnight at 4° C.Culture supernatants were diluted to the desired level in 10 mM sodiumphosphate buffer (pH 8.0), containing 0.5 M sodium chloride and 0.1%Tween-20. Coated plates were incubated with diluted culture supernatantsfrom the cell line, washed, incubated with peroxidase-labeled goatantibodies against human IgG and IgM and developed (15 min) by additionof 100 μl of the chromogenic substrate o-phenylaenediamine (OPD)(Sigma™). The reaction was stopped after 30 min by adding 1 M sulphuricacid, and the absorbances were read at 490 nm (FIG. 1).

Example 2. Inhibition of TNF Release by Anti-HMGB1 Monoclonal Antibodies

The ability of particular human mAbs to inhibit HMGB1-induced TNFrelease was assessed in human primary monocytes administered only HMGB1,HMGB1 plus particular HMGB1 mAbs, or an isotype control antibody.Freshly purified human monocytes cultures were stimulated with 1 μg/mlpurified bovine HMGB1 and culture supernatant were sampled four hourslater to be assayed for TNF. As depicted in FIG. 2, two particular humanmAbs inhibited TNF release, indicating that such antibodies could beused to neutralize the biological activity of HMGB1 and thus foralleviating neuropathic diseases.

Example 3. Completion of mAb Binding to HMGB1 by the Immunogen

As shown in FIG. 3, QCM was used to perform kinetic analysis ofinteraction between HMGB1, a representing mAb and peptides with the sameamino composition. HMGB1 was injected to saturate the chip. Thefrequency change of mAb with immunogen-containing peptide or anirrelevant peptide with the same amino composition was analyzed by Aqua™software. The results indicated that the mAb binding to HMGB1 can beonly replaced by the immunogen-containing peptide.

Example 4. HMGB1 Neutralization In Vivo

Chronic experimental allergic encephalomyelitis (EAE), which representsan animal model of human neuromyelitis, was induced in C57BL/6 mice byimmunization with an emulsion of MOG35-55 or MOG1-125 in completeFreund's adjuvant (CFA) distributed in three spots on the flank,followed by administration of pertussis toxin (PTX) in PBS, first on theday of immunization and then again the following day. EAE is scored onscale 0 to 5, based from no obvious changes in motor function comparedto non-immunized mice to mice spontaneously rolling in the cage,respectively. 100 μs of purified anti-HMGB1 antibody or isotype controlantibody was injected intraperitoneally at days 5 and 7 post MOGinjection. From day 15, Human anti-HMGB1 mAb demonstrated significantalleviating effect (FIG. 4).

Although the present invention has been explained in relation to itspreferred embodiment, it is to be understood that many other possiblemodifications and variations can be made without departing from thespirit and scope of the invention as hereinafter claimed.

What is claimed is:
 1. A method for producing human monoclonalantibodies that binds to HMGB1, comprising the following steps of: (a)providing a synthetic antigen consisting of T-helper epitope SEQ ID NO:2 coupled to SEQ ID NO: 3 to immunize a group of human CD56⁻lymphocytes; (b) the lymphocytes immunized by the synthetic antigen areenriched by antigen-coupled magnetic beads; (c) adding Epstein-Barrvirus (EBV) to the human CD56⁻ lymphocytes immunized by the syntheticantigen; and (d) selecting the EBV-infected cells that produce theantibodies that recognize HMGB1.